Use of adenylate cyclase or bacteria producing it as vaccines against bordetella

ABSTRACT

The invention provides for the utilisation of a bacterial vaccining preparation elaborated from a given bacterium of the genus Bordetella or an adenyl cyclase in order to prepare an antigen for the protection of human beings or animals against infections and the toxic effects caused by a bacterium of the genus Bordetella but different from that of the vaccining preparation.

[0001] The invention relates to vaccines capable of protecting man oranimals against lethal infections caused by the Bordetella. It relates,in particular, to the use of vaccinating preparations developed fromBordetella or more particularly from adenylate cyclase produced by thesebacteria as protective antigens against the toxic effects of theinfections due to Bordetella.

[0002] It is known that the Bordetella, more particularly Bordetellapertussis, Bordetella parapertussis and Bordetella bronchiseptica, areresponsible for respiratory diseases in the vertebrates.

[0003] Thus, in man, B.pertussis is responsible for whooping cough, aninfantile disease very wide spread throughout the world.

[0004] The vaccination against whooping cough has hitherto been mostusually carried out with the aid of inactivated whole bacteria.

[0005] However, such vaccines are not always devoid of toxicity in viewof the fact that the virulence factors are constituted by proteinssecreted by the bacteria and not by the bacteria themselves. Theproteins can thus exert serious pathological effects, even after thedeath of the bacteria.

[0006] Among the determinants of virulence of B.pertussis, mentionshould be made of the adhesins such as the agglutinogens (AGG), thefilamentous hemagglutinin (FHA) and the pertussis toxin (PTx), acytotracheal toxin (CTT), a dermonecrotic toxin (DNT) and an adenylatecyclase-hemolysin (AC). The latter is synthesized in the form of a largeprecursor of 1706 residues. The amino-terminal part of the moleculebears the adenylate cyclase activity and the carboxy-terminal partpossesses a strong homology with the Hly gene product of E.coli(hemolysin). This adenylate cyclase has the property of being activatedby calmodulin.

[0007] The research for more suitable methods of prevention has led theinventors to study the role of each of the determinants of virulence andto develop an experimental model of infection mimicking the process ofthe natural disease.

[0008] These studies have made it possible to note the role of adenylatecyclase as cytotoxin and as protective antigen, and to develop a noveluse of the vaccinating preparations based on adenylate cyclase orbacterial preparations producing adenylate cyclase.

[0009] The invention relates more particularly to the use of a bacterialvaccinating preparation developed from a given bacterium of theBordetella genus, this use being characterized in that it is used toprotect man or animals against the infections and the toxic effectscaused by a bacterium of the Bordetella genus but different from that ofthe vaccinating preparation.

[0010] In accordance with another feature, the invention also relates tothe use of a vaccinating preparation developed from an adenylate cyclaseof a given bacterium of the Bordetella genus in order to protect man oranimals against the infections and the toxic effects caused by abacterium of the Bordetella genus different from that from which theadenylate cyclase is derived.

[0011] The bacteria of the Bordetella genus are selected fromB.pertussis, B.parapertussis, and B.bronchiseptica.

[0012] In a preferred embodiment of the invention, the vaccinatingpreparation is developed from B.bronchiseptica or from the adenylatecyclase of B.bronchiseptica and is used to provide protection againstthe infections and the toxic effects caused by B.pertussis orB.parapertussis.

[0013] It should be noted that this crossed protection obtained startingfrom strains of B.bronchiseptica offers the advantage of using strainswith more rapid growth than B.pertussis or B.parapertussis in order todevelop vaccines against the infections caused by B.pertussis orB.parapertussis.

[0014] According to another embodiment of the invention, the vaccinatingpreparation is developed from B.pertussis or the adenylate cyclase ofB.pertussis and is used to provide protection against the infections andthe toxic effects caused by B.bronchiseptica or B.parapertussis.

[0015] According to yet another embodiment of the invention, thevaccinating preparation used is developed from B.parapertussis or theadenylate cyclase of B.parapertussis and is used to provide protectionagainst the infections and the toxic effects caused by B.pertussis or B.bronchiseptica.

[0016] According to yet another embodiment of the invention, thevaccinating preparation used is developed from B.parapertussis or theadenylate cyclase of B.parapertussis and is used to provide protectionagainst the infections and the toxic effects caused by B.pertussis orB.bronchiseptica.

[0017] The adenylate cyclase of the Bordetella to which reference hasbeen made above is an adenylate cyclase such as that obtained by placinga supernatant of a bacterial culture of Bordetella in contact with anAffigel-calmodulin® gel.

[0018] More particularly, it is a preparation of adenylate cyclase suchas those described in the application FR 2606789 filed on 17.11.86

[0019] It will be recalled that these preparations are characterized inthat they possess a high purity and are almost completely devoid ofcontaminating bacterial products, in particular pertussis toxins,lipopolysaccharide (or LPS) and filamentous hemagglutinin (or FHA).

[0020] The adenylate cyclase of these preparations exists in ahomogeneous form sedimenting on a sucrose density gradient with acoefficient S equal to 3.6. It exists in two structurally related,molecular forms of 45 and 43 kDa respectively.

[0021] Such preparations of adenylate cyclase possess an activity whichmay attain and even exceed 1600 μmole of cAMP min⁻¹ mg⁻¹.

[0022] Such preparations may be prepared from bacterial culturesexpressing the AC (adenylate cyclase), more particularly from pathogenicbacteria whose AC is capable of interfering with the AC of eucaryoticcells, by placing a supernatant of bacterial cultures expressing theadenylate cyclase and concentrated beforehand or an extract of thesebacteria in contact with calmodulin.

[0023] In order to obtain the enzyme in the free form, calmodulin fixedto a support is used, then the absorbed enzyme is recovered with the aidof a denaturing agent which is removed in turn, and the preparation ofthe free enzyme is recovered.

[0024] The support is more particularly constituted by a material inertwith respect to the preparation containing the enzyme, and is capable ofretaining molecules of high molecular weight, such as a gel or afiltering material.

[0025] The filtering material is advantageously made of nitro-celluloseor a plastic material and exhibits a porosity of 0.45 -0.22 μm.

[0026] The denaturing agent is preferably urea and preferably 4 to 8.8M.

[0027] The concentrated supernatant of the bacterial culture is obtainedby subjecting a supernatant of bacterial cultures expressing the AC toone or several filtration steps with the aid of nitrocellulose filtersor filters of plastic material of porosity advantageously from 0.45 to0.22 μm, then by incubating the filters with a detergent in order torelease the AC and by removing from the AC preparation the insolublematerials present.

[0028] The detergent is, for example, Triton® or NP40®.

[0029] The bacterial extract is obtained by treatment of bacterial cellsexpressing the adenylate cyclase with urea and recovery of thesupernatant.

[0030] As a variant, an adenylate cyclase is used such as that expressedby the nucleotide sequence given in the only figure with thecorresponding amino acid sequence.

[0031] It will be obvious that the bases of the nucleotide sequenceunder consideration may be in an order different from that found in thegenes and/or that these bases may be, where appropriate, substitutedprovided that a probe developed from such a sequence gives acharacteristic and unequivocal response with regard to the capactity torecognize the presence of a gene coding for a protein with adenylatecyclase activity.

[0032] Any nucleotide sequence which can hybridize with this chainsequence such as that obtainable by reverse enzymatic transcription ofthe corresponding RNA or also by chemical synthesis is also included inthe framework of the invention.

[0033] The above vaccinating preparations used may or may not becombined with the FHA and/or the PTx in the same inoculum.

[0034] In this latter case, the FHA is administered at the same time asthe vaccinating preparation or at a different time. The preparations ofFHA are advantageously obtained, for example, according to the method ofSATO et al in Infect. Immun., 1983, 41, 310-320 or that of IMAIZUMI etal in Journal of Microbiol. Methods, 2, 334-347 (1984).

[0035] In accordance with another feature, the invention relates tovaccines capable of inducing a protection against the toxic effects andthe infections caused by the Bordetella, molecular vaccines containingat least the active part of the amino acid sequence represented in theonly figure, where appropiate, combined or not in the same inoculum withthe FHA and/or the PTx.

[0036] These vaccines are advantageously used in conformity with theinvention in order to carry out crossed protections.

[0037] In the implementation of the invention, the vaccinatingpreparations based on bacteria or the adenylate cyclase, advantageouslypurified, produced by these bacteria as well as the vaccines developedfrom recombinant adenylate cyclase are used at the usual doses and inthe usual forms of administration, in particular in the standard formswhich can be administered by the intranasal, oral or parenteral routes.

[0038] Other characteristics and advantages of the invention will becomeapparent in the description of the examples which follow and byreferring to the only figure which shows the nucleotide sequence of theactive part of the gene of B.pertussis coding for the adenylate cyclaseand the corresponding sequence of amino acids.

[0039] The model of respiratory infection used in the assays reported inthe examples is a model of infection by the intranasal route. The invivo selection from a mouse lung infected by B.pertussis of ahyperpathogenic derivative of the virulent strain of B.pertussis 18323S(international reference strain for the evaluation tests of vaccines)has made it possible to observe in the adult mouse, after injection ofbacteria by the intranasal route, an acute hemorrhagic edematousalveolitis (AHEA), lethal within 48-72 h.

[0040] The analyses of the products of the examples are performed asfollows.

[0041] The activity of the AC is measured according to the method ofWhite A. A. in Methods Enzymol., 38C, 41-46, 1974, as modified byHanoune et al (J. Biol. Chem., 252, 2039-2046, 1977).

[0042] The activities of PTx and FHA are measured according to themethod of TUOMANON S. and WEISS A. in J. Inf. Dis., 152, 118-125, 1985.

EXAMPLE 1 CULTURE OF B.BRONCHISEPTICA AND ISOLATION OF A SUPERNATANTWITH A HIGH CONCENTRATION OF ADENYLATE CYCLASE

[0043] A preculture of B.bronchiseptica 9.73, phase I and aspontaneously avirulent, stable variant of this strain, phase IV aregrown for 48 hours at 36° C. on a modified Stainer-Scholte agar mediumsupplemented with cyclodextrin (according to IMAIZUIMI et al in J. Chim.Microbiol., 17, 781-786, 1983). The preculture is then transferred to amodified Stainer-Scholte liquid medium without cyclodextrin. TheB.bronchiseptica 9.73 strain was isolated from the nostrils of a hare.

[0044] The liquid cultures are stirred at 150 rev./min. for 15 hours at36° C. in a 1 liter Erlenmeyer containing 250 ml of medium. The cultureis continued until an optical density OD₆₅₀=1.2±0.2 is obtained. Thebacteria are then removed by centrifugation at 5000×g for 25 min.

[0045] The culture supernatants are stored at −30° C. until used or areconcentrated immediately.

[0046] For the purposes of concentration, 3 liters of culturesupernatant containing about 10 μg of proteins and 0.1 unit of enzymeper ml are filtered on filters of the Millipore® HAWP type of 0.45 μm.In this way, more than 90% of the enzymatic activity is retained on thefilters. About 80% of this activity can be recuperated by incubation ofthe filters in 40 ml of a buffer A constituted by 50 mM Tris.HCl, pH 8containing 6 mM of MgCl₂ and 0.1% of Triton X.100®. The insolublematerial is removed by centrifugation for 30 min. at 15000×g at 4° C.The specific activity of the concentrated culture supernatant is 115units per milligram of protein.

[0047] A bacterial suspension of the strain 9.73 was injected into amouse by the intranasal route; 48 hours later the lungs were excised andground and a hemolytic clone was isolated, namely the clone 9.73S. Thisclone was deposited on May 12, 1989 with the CNCM under the No. I-858.This clone is hemolytic and synthesizes large amounts of adenylatecyclase. A bacterial suspension of this clone causes hemorrhagic edemain the lung of the mouse.

EXAMPLE 2 PROCEDURE FOR THE ISOLATION OF PURIFIED EXTRA-CYTOPLASMICADENYLATE CYCLASE IN THE FREE FORM

[0048] 40 ml of concentrated culture supernatant are added to 0.8 to 1ml of Affigel-calmodulin® and the mixture is stirred slowly at 4° C. for18 hours. More than ⅔ of the enzymatic activity is retained on the gel.

[0049] The Affigel-calmodulin is sedimented by centrifugation at 300×gfor 1 minute, it is washed several times with 0.5 M NaCl in buffer A.The adenylate cyclase is recovered from the gel with the aid of 2.5 mlof 8.8M urea in buffer A. The urea is removed by filtration on a columnof Sephadex G-25®, equilibrated with buffer A.

[0050] This enzymatic preparation possesses a specific activity of 1100units/mg of protein.

[0051] It may be stored at −80° C. without loss of activity for severalweeks.

EXAMPLE 3 PROCEDURE FOR THE ISOLATION OF PURIFIED ADENYLATE CYCLASE FROMBACTERIAL EXTRACTS

[0052] A bacterial culture expressing AC activatable by calmodulin issubjected to a treatment with 8M urea for about 2 hours at roomtemperature. The supernatant which constitutes the bacterial extract isrecovered and it is placed in contact with the Affigel-calmodulin gelunder the conditions mentioned above.

[0053] An enzymatic preparation of high purity is obtained exhibitingapproximately the same specific activity.

EXAMPLE 4 STUDY OF THE SECRETION OF AC, PTx and FHA, AND THE LD50 OFBORDETELLA STRAINS

[0054] In table 1 below, are presented the results obtained bydetermining the factors of virulence AC, PTx and FHA and by measuringthe LD50 (dose killing 50% of the mice in a batch) of different strainsof Bordetella.

[0055] They are strains of B.pertussis (BP), B.parapertussis (BPP) andB.bronchiseptica (BB) corresponding either to hyperpathogenic strains,which secrete large amounts of adenylate cyclase (these strains bear thereference S in the table), or to parental strains of the hyperpathogenicstrains. TABLE 1 AC PTx FHA LD 50 BP18323 20 1  2 3 10⁸ BP18323S 180 4 2 10⁷ BP8144 2 (+) BP8144S 6 (+) BP8132 1 (+) 32 3.2 10⁸ BP8132S 3 (+)32 <1,2 10⁸ BPP632 8 — 64 2 10⁸ BPP632S 35 — 64 <10⁸ BB973S 110 — 16 410⁸

[0056] These results reveal the high production of adenylate cyclase byhypopathogenic strains, more particularly by B.bronchiseptica 973S andB.pertussis 18323S.

[0057] The secretion of PTx is also increased in the hyperpathogenicderivatives as the results relating to B.pertussis show whereas thesecretion of FHA remains the same in the two strains.

[0058]B.bronchiseptica and B.parapertussis, which synthesize the samefactors as B.pertussis with the exception of the PTx, induce ahemorrhagic edematous alveolitis in the mouse similar to that induced byB.pertussis. On the other hand, a strain derived from the virulentstrain B.pertussis 8132, the strain B.P.348 accommodating a transposonTn5 in the structural gene of the AC, inactivating the latter, isincapable of inducing a AHEA in the mouse. Nonetheless, this mutantsecretes all of the other factors of virulence, in particular the PTx.

[0059] The body of these results designates the AC as the factorresponsible for the hemorrhagic edematous alveolitis in the mouse.

EXAMPLE 5 1. EXPERIMENTS OF ACTIVE PROTECTION STARTING FROM BACTERIALVACCINES

[0060] Protection experiments (against B.pertussis) were carried outwith the aid of different bacterial vaccines (usually 3 sub-cutaneousinjections of 250 μl of a bacterial suspension containing 10⁹bacteria/ml, heated to 56° C. at weekly intervals).

[0061] In table 2 below are reported the results obtained by usingstrains of B.bronchiseptica 9.73S as well as strains of B.pertussis18323S, B.parapertussis 63.25 and B.avium (Blike Hewouet) for thedevelopment of the bacterial vaccines.

[0062] The B.pertussis strain is administered two weeks after the lastimmunization: 50 μl of 10⁸ live bacteria of the B.pertussis 18323Sstrain are injected by the intranasal route. LETHAL TEST STRAIN INJECTED(B.pertussis) 0 10/10  B.bronchlseptica 0/10 B.pertussis 0/10B.paraportussis 3/10 B.avium 9/10

[0063] It will be noted that the mice vaccinated with the aid ofpreparations of B.bronchiseptica are completely protected against thelethal test with B.pertussis (crossed protection), like those vaccinatedwith B.pertussis.

[0064] On the other hand, the mice vaccinated with preparations ofB.avium are not protected, which works in favour of the role of the ACin the induction of hemorrhagic edematous alveolitis.

2. EXPERIMENTS OF ACTIVE PROTECTION STARTING FROM PURIFIED ANTIGENS

[0065] Purified antigens are used.

[0066] The preparations of AC are purified either from the culturesupernatant (70% of the enzyme is excreted by the bacterium), or fromthe bacteria by working advantageously according to the procedure of thepatent application FR 2606789 of 17.11.86.

[0067] Mice were immunized with different doses of antigen (3sub-cutaneous injections of 250 μl of antigen in 10 mM Tris buffer, pH7, containing 1 mg/ml of Am⁺⁺⁺ at weekly intervals) before the lethaltest with B.pertussis was made.

[0068] For the lethal test, 50 μl of a bacterial suspension of thevirulent and hyperpathogenic strain 18323S is injected by the intranasalroute 2 weeks after the last immunization. The bacterial concentrationof the suspension is 10⁸ in test a, 2×10⁸ in test b, and 10⁷ in test c.

[0069] In table 3 below, the results are reported of the protectionobtained against a strain of B.pertussis (BP 18323S) at differentconcentrations after injection of AC, recombinant AC of a vaccine, PTxand FHA.

[0070] The recombinant AC is advantageously such as described in thepatent application FR 2621597 of 24.7.87. It is a fragment correspondingto the structural gene of the AC which could be expressed in E.coli.This fragment, which bears the adenylate cyclase activity and thebinding site for calmodulin, was purified to homogeneity (see thefigure). AC AC_(rec) VACCIN PTX FHA 0 a: lethal test B.P. 18323S 10/LD503/0.9 μg 3/1.5 μg 3/10°b 2/2 μg  2/10 μg 9/12 1/10 0/40 9/22 20/22 20/22b: lethal test B.P. 18323S 20/LD50 3/2 μg 3/10°b 5/10 3/10 10/10 c:lethal test B.P. 18323S 1/LD50 3/6 μg 3/6 μg 3/18 μg 3/2 μg 3/10 μg 0/100/10 0/10  5/10  5/10

[0071] The results of lethality are expressed as the number of dead miceover the total number of mice.

[0072] The results obtained show the production of a synthesis ofanti-AC antibodies and a protection of the mice after immunization withAC (three immunizations of 1.5 μl of AC). This protection is almostcomplete when the lethal test is performed with a number of bacteriaequal to (10⁷ live bacteria) or 10 times higher than the lethal dose 50(10⁸ live bacteria); it is 60% when the lethal test is performed with anumber of bacteria equal to 20 times the lethal dose 50 (2.10⁸ lethalbacteria).

[0073] Complete protection was obtained after immunization of mice withthe recombinant AC when the lethal test was performed with a number ofbacteria equal to the lethal dose 50. The immunizations performed withFHA did not show a protective effect of this toxin in spite of thepresence of antibodies in the mice immunized even when the lethal testwas performed with a small number of bacteria.

[0074] The experiments conducted with the PTx show a partial protectionafter two immunizations with 2 μg of PTx when the lethal test is 10times higher than the lethal dose 50 but it must be pointed out that, inthis case, the synthesis of anti-PTx antibodies is very low. When themice are immunized with 3 times 2 μg of PTx, the synthesis of antibodiesis much greater and we obtain complete protection when the lethal testis performed with a number of bacteria equal to the lethal dose 50.

[0075] In sum, these results show that the adenylate cyclase constitutesthe cytotoxin responsible for the hemorrhagic edematous alveolitisobserved in the mouse infected by B.pertussis and constitutes an antigenprotecting against such lesions. The PTx and the FHA would appear toplay no role in the induction of this AHEA but could be protectiveantigens of such lesions by inhibiting the adhesion of bacteria to therespiratory epithelium.

1. Use of a bacterial vaccinating preparation developed from a givenbacterium of the Bordetella genus for the preparation of a protectiveantigen for man and animals against the infections and the toxic effectscaused by a bacterium of the Bordetella genus but different from that ofthe vaccinating preparation.
 2. Use of a vaccinating preparationdeveloped from an adenylate cyclase of a given bacterium of theBordetella genus for the preparation of a protective antigen for man oranimals against the infections and the toxic effects caused by abacterium of the Bordetella genus different from that from which theadenylate cyclase is derived, the said adenylate cyclase being such asthat obtained by placing the supernatant of a bacterial culture ofBordetella in contact with a Affigel-calmodulin® gel.
 3. Use accordingto claim 1 or 2, characterized in that the bacteria of the Bordetellagenus are selected from B.pertussis, B.parapertussis, andB.bronchiseptica.
 4. Use according to claim 3, characterized in that thevaccinating preparation is developed from B.bronchiseptica or from theadenylate cyclase of B.bronchiseptica and that it is used in order toprepare a protective antigen against the infections and the toxiceffects caused by B.pertussis or B.parapertussis, the said adenylatecyclase being such as that obtained by placing the supernatant of abacterial culture of Bordetella in contact with a Affigel-calmodulin®gel.
 5. Use according to claim 3, characterized in that the vaccinatingpreparation is developed from B.pertussis or the adenylate cyclase ofB.pertussis and that it is used in order to prepare a protective antigenagainst the infections and the toxic effects caused by B.bronchisepticaor B.parapertussis, the said adenylate cyclase being such as thatobtained by placing the supernatant of a bacterial culture of Bordetellain contact with a Affigel-calmodulin® gel.
 6. Use according to claim 3,characterized in that the vaccinating preparation is developed fromB.parapertussis or the adenylate cyclase of B.parapertussis and that itis used in order to prepare a protective antigen against the infectionsand the toxic effects caused by B.pertussis or B.bronchiseptica, thesaid adenylate cyclase being such as that obtained by placing thesupernatant of a bacterial culture of Bordetella in contact with aAffigel-calmodulin® gel.
 7. Vaccines capable of inducing protectionagainst the toxic effects and the infections caused by the Bordetella,characterized in that they are molecular vaccines containing at leastthe active part of the amino acid sequence represented in the figure,where appropriate combined or not, in the same inoculum, with FHA and/orPTx.
 8. Use according to any one of the claims 1 to 6, characterized inthat the vaccinating preparations are combined or not, in the sameinoculum, with FHA and/or PTx.